Xylose reductase XR21 mutant and application thereof

Abstract

The invention relates to the technical field of enzyme engineering, in particular to a xylose reductase XR21 mutant and application thereof. Specifically, the invention provides a novel xylose reductase mutant aiming at the defect of poorer thermal stability of the existing wild type xylose reductase, and particularly provides a xylose reductase mutant which is obtained by performing single or multiple site amino acid mutation on the 19th site, the 48th site, the 79th site, the 208th site and the 300th site of a xylose reductase sequence shown as SEQ ID NO: 2, compared with wild type xylose reductase, the obtained xylose reductase mutant has the advantages that the activity of catalyzing an epimer substrate arabinose into arabitol can be obviously reduced, and meanwhile, the selectivity to xylose is improved, so that the operation process of downstream separation and purification is simplified, and the biological catalysis requirement of green industry is met.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to the XR21 mutant of xylose reductase and its applications. Background Technology

[0002] Xylitol is a pentose sugar alcohol that is naturally found in fruits and vegetables (such as strawberries and spinach). It has a similar sweetness to sucrose (about 95% of sucrose) but is low in calories (2.4 kcal / g, compared to 4 kcal / g for sucrose) and its metabolism does not depend on insulin. Therefore, it can be widely used in food, medical, and chemical industries as a diabetes-friendly sweetener, a sucrose substitute, and a biodegradable material for producing xylitol-based polyesters.

[0003] Currently, the main methods for preparing xylitol include: Traditional chemical processes primarily use lignocellulose (such as corn cobs and sugarcane bagasse) as raw material, which is acid-hydrolyzed to produce xylose, and then catalytically hydrogenated to xylitol using Ni / Al2O3 as a catalyst under high temperature and pressure. Although this method is mature, lignocellulose contains multiple components such as xylose, glucose, and arabinose, and the Ni / Al2O3 catalyst lacks selectivity during catalysis, thus generating other sugar alcohols during the catalytic process. To obtain high-purity xylitol, pretreatment is required to separate and purify hemicellulose to obtain xylose or to further purify the sugar alcohol product, which is complex and costly. Furthermore, nickel, as a heavy metal catalyst, can cause serious pollution. Emerging green processes, such as microbial catalysis, utilize genetically engineered microorganisms (such as yeast)... Candida tropicalis ,bacteria Escherichia coli This method expresses xylose reductase and xylitol dehydrogenase to convert xylose into xylitol. This approach effectively improves the selectivity of xylitol catalysis and avoids heavy metal catalyst contamination. While xylose reductase catalysis for xylitol production has been reported, natural xylose reductase lacks substrate specificity, and the catalytic process produces the byproduct arabinitol, increasing the difficulty of xylitol separation and purification, severely limiting the industrial application of xylose reductase.

[0004] Although most natural xylose reductases exhibit poor substrate selectivity, studies have shown that their selectivity can be successfully improved through molecular modification techniques. Nair et al. [reference needed - likely referring to a specific enzyme or process] extracted from *Neurospora crassa* (…). Neurospora crassaA xylose reductase was isolated from [a specific organism] and subjected to directed evolution, resulting in a mutant (VMQCI) with a 50-fold increased selectivity for arabinose. During resting cell transformation, the arabinitol content was significantly reduced. To further improve the selectivity of xylose reductase, its conserved coenzyme binding site was modified, and a new mutant enzyme, VMQCIRTI, was screened. Its xylose selectivity was approximately 12-fold higher than that of the wild type, and its activity was approximately 2.22-fold higher than that of the mutant enzyme obtained by Niar et al.

[0005] Based on existing findings, and combining bioinformatics analysis with structural biology techniques, we will conduct in-depth research on the substrate recognition mechanism of xylose reductase and perform semi-rational design of xylose reductase. This will hopefully lead to the development of novel xylose reductase mutants with higher xylose selectivity, which is of great significance for achieving green and efficient production of xylitol. Summary of the Invention

[0006] This invention addresses the low selectivity of existing xylose reductases by providing a novel xylose reductase mutant. Specifically, it is obtained by mutating one or more amino acids selected from positions 19, 48, 79, 213, and 305 of the xylose reductase sequence shown in SEQ ID NO: 2. The resulting xylose reductase mutant exhibits significantly reduced catalytic activity towards the epimeric substrate arabinose to arabinitol compared to the original xylose reductase, while simultaneously improving selectivity for xylose. Its advantages include simplified downstream separation and purification processes, meeting the requirements of green industrial biocatalysis.

[0007] In this regard, the present invention includes, but is not limited to, the following technical solutions: In one aspect, the present invention provides a xylose reductase mutant, characterized in that the mutant has 5 or fewer substitutions relative to SEQ ID NO: 2 and is similar to SEQ ID NO: 2. The sequence identity is less than 100%, wherein the substitution is selected from: F19A (phenylalanine at position 19 is mutated to alanine), F19L (phenylalanine at position 19 is mutated to leucine), F19I (phenylalanine at position 19 is mutated to isoleucine), F19M (phenylalanine at position 19 is mutated to methionine), F19G (phenylalanine at position 19 is mutated to glycine), F19S (phenylalanine at position 19 is mutated to serine), F19T (phenylalanine at position 19 is mutated to threonine), F19Y (phenylalanine at position 19 is mutated to tyrosine), F19H (phenylalanine at position 19 is mutated to histidine), F19E (phenylalanine at position 19 is mutated to glutamic acid), C48A (cysteine ​​at position 48 is mutated to alanine), C48V (cysteine ​​at position 48 is mutated to valine), C48S (cysteine ​​at position 48 is mutated to serine), K79A (… (Lysine at position 79 is mutated to alanine), K79M (Lysine at position 79 is mutated to methionine), K79N (Lysine at position 79 is mutated to asparagine), K79R (Lysine at position 79 is mutated to arginine), A208L (Lysine at position 208 is mutated to leucine), A208W (Lysine at position 208 is mutated to tryptophan), A208G (Lysine at position 208 is mutated to glycine), A208Y (Lysine at position 208 is mutated to leucine). A208D (alanine at position 208 is replaced by aspartic acid), R300M (arginine at position 300 is replaced by methionine), R300T (arginine at position 300 is replaced by threonine), R300C (arginine at position 300 is replaced by cysteine), R300Q (arginine at position 300 is replaced by glutamine), and R300E (arginine at position 300 is replaced by glutamic acid), or combinations thereof.

[0008] In one aspect, the substitutes described in this invention are selected from: F19G, C48V, K79A, A208L and R300C, or combinations thereof.

[0009] In another aspect, the present invention provides a xylose reductase mutant obtained by performing the following mutation on SEQ ID NO: 2: F19G; C48V; K79A; A208L; R300C; F19G+K79A; C48V+A208L; A208L+R300C; F19G+C48V+A208L; F19G+K79A+R300C; C48V+K79A+R300C; F19G+C48V+K79A+A208L; C48V+K79A+A208L+R300C; F19G+C48V+K79A+A208L+R300C; F19A; F19L; F19I; F19M; F19S; F19T; F19Y; F19H; F19E; C48A; C48S; K79M; K79N; K79R; A208W; A208G; A208Y; A208D; R300M; R300T; R300Q or R300E.

[0010] In another aspect, the present invention provides a xylose reductase mutant obtained by performing the following mutation on SEQ ID NO: 2: F19G; C48V; K79A; A208L; R300C; F19G+K79A; C48V+A208L; A208L+R300C; F19G+C48V+A208L; F19G+K79A+R300C; C48V+K79A+R300C; F19G+C48V+K79A+A208L; C48V+K79A+A208L+R300C; or F19G+C48V+K79A+A208L+R300C.

[0011] In another aspect, the present invention provides a xylose reductase mutant obtained by performing the following mutation on SEQ ID NO: 2: F19G; A208L; F19G+K79A; C48V+A208L; A208L+R300C; F19G+C48V+A208L; F19G+K79A+R300C; C48V+K79A+R300C; F19G+C48V+K79A+A208L; C48V+K79A+A208L+R300C; or F19G+C48V+K79A+A208L+R300C.

[0012] In the technical solution of the present invention concerning xylose reductase mutants, "+" represents the simultaneous presence of two or more mutation forms. For example, F19G+K79A represents a xylose reductase mutant obtained by mutating the xylose reductase sequence shown in SEQ ID NO: 2 as follows: F19G and K79A; F19G+C48V+K79A+A208L+R300C represents a xylose reductase mutant obtained by mutating the xylose reductase sequence shown in SEQ ID NO: 2 as follows: F19G, C48V, K79A, A208L, and R300C.

[0013] In another aspect, the present invention provides a polynucleotide encoding the xylose reductase mutant described in the present invention.

[0014] In another aspect, the present invention provides a recombinant vector comprising the polynucleotides described herein.

[0015] In one aspect, the vector described in this invention is pET-28a. The expression vector of this invention may also be replaced with other suitable expression vectors conventionally used in the art.

[0016] In another aspect, the present invention provides a host cell characterized in that it comprises the polynucleotide or the recombinant vector described in the present invention.

[0017] In one aspect, the host cell described in this invention is a fungal cell, a bacterial cell, or a plant cell. Preferably, the host cell is a bacterial cell, and more preferably, the bacterial cell is an *Escherichia coli* cell. It should be noted that the plant cell described in this invention is not intended to protect any specific plant, and this invention does not disclose any method for developing plant cells into a complete plant. The plant cell described in this invention is only used as an engineered cell for expressing the enzyme mutant of this invention.

[0018] In one aspect, the host cell of the present invention is a bacterial cell, preferably, the bacterial cell is an *Escherichia coli* cell. More preferably, the host cell is *Escherichia coli* (…). E. coli BL21(DE3) cells.

[0019] In one aspect, the Escherichia coli cells of the present invention are E. coli BL21(DE3) cells.

[0020] In another aspect, the present invention provides the use of the xylose reductase mutant, polynucleotide, recombinant vector or host cell described herein in the production of xylitol.

[0021] In one aspect, the present invention also provides a method for preparing xylitol, the method comprising: mixing the xylose reductase mutant of the present invention or the host cell of the present invention with xylose and NADPH under suitable conditions to generate xylitol.

[0022] Those skilled in the art can routinely confirm the suitable conditions for expressing the xylose reductase mutant and the suitable conditions for generating xylitol as described in this invention.

[0023] In one aspect, xylose is used as a substrate, and crude enzyme solution obtained by inducing expression of recombinant engineered bacteria containing xylose reductase mutant encoding gene is used as a catalyst to catalyze the reduction reaction to produce xylitol.

[0024] In one aspect, using xylose as a substrate, recombinant engineered bacteria containing a gene encoding a xylose reductase mutant are added to catalyze the production of xylitol from xylose under suitable conditions.

[0025] In one aspect, the system for the reduction reaction to produce xylitol also includes a coenzyme cycle; said coenzyme cycle includes a coenzyme, glucose, and glucose dehydrogenase, wherein the coenzyme includes NADP. + And NADPH.

[0026] In one respect, the concentration of the substrate xylose is 50–800 mM.

[0027] In another aspect, the present invention also provides a method for preparing xylitol, using xylose as a substrate, NADPH as a coenzyme, and the xylose reductase mutant described in this invention as a catalyst to catalyze a reduction reaction to produce xylitol. The cells expressing the xylose reductase mutant can be the host cells described in this invention.

[0028] In another aspect, the present invention also provides a method for preparing xylitol, using a mixed lignin hydrolysate as a substrate and a recombinant engineered bacterium containing a xylose reductase mutant encoding gene as a whole-cell catalyst to catalyze a reduction reaction to produce xylitol or arabinitol.

[0029] In one aspect, the reaction system containing a whole-cell catalyst contains glucose. Preferably, the concentration of glucose is 10 g / L.

[0030] In one aspect, the reaction of the present invention is carried out under conditions of pH 6 to 9 (preferably pH 7.5).

[0031] In one aspect, the reaction temperature is controlled at 20-60°C, preferably 20-50°C, and more preferably 30°C.

[0032] In one aspect, the concentration of the substrate xylose is 50-800 mM.

[0033] In one respect, the concentration of lignin hydrolysate as substrate is 10-20 g / L.

[0034] In one aspect, the amount of xylose reductase mutant catalyst used in the reaction system was 20-80 μL, and the substrate concentration was 50 mM.

[0035] In one aspect, the amount of recombinant engineered bacteria containing the xylose reductase mutant encoding gene in the reaction system was 5 g / L.

[0036] In one aspect, the catalyst used in the reaction system is the crude enzyme solution after cell disruption.

[0037] In another aspect, the present invention provides a method for producing the xylose reductase mutant described herein, comprising the following steps: (1) The host cells of the present invention are cultured under suitable conditions for expressing the xylose reductase mutant; and (2) The xylose reductase mutant was recovered.

[0038] In one aspect, the preparation method of xylose reductase mutant bacterial cells is as follows: Engineered bacteria containing the xylose reductase mutant encoding gene are inoculated into liquid LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking for 12 h. Subsequently, they are transferred at an inoculum volume of 2% (v / v) into fresh liquid LB medium containing 50 μg / mL kanamycin and cultured at 37°C with shaking until the bacterial cell concentration reaches OD0.05. 600 The initial concentration was 0.4-0.8, followed by the addition of IPTG to a final concentration of 0.5 mM, and induction culture was carried out at 18°C ​​for 16 h. The fermentation broth was centrifuged at 4000 rpm for 10 min, and the cells were collected.

[0039] In this invention, the source is Penicillium daleae The base sequence of xylose reductase has been codon-optimized for the expression host Escherichia coli, resulting in the nucleotide sequence shown in SEQ ID NO: 1, whose encoded amino acid sequence is shown in SEQ ID NO: 2.

[0040] SEQ ID NO: 1: ATGTCTTCTCCGGTTGTTAAACTGTCTTCTGGTTACGAAATGCCGCTGGTTGGTTTCGGTCTGTGGAAAGTTAACAACGATACCTGCGCGGATCAGGTTTACTCTGCGATCAAAGCGGGTTACCGTCTGTTCGATGGTGCGTGCGATTACGGTAACGAAGTTGAAGTTGGTCAGGGTGTTGCGCGTGCGATTAAAGAAGGTATCGTTAAACGTGAAGATCTGTTCCTGGTTTCTAAACTGTGGAACTCTTTCCACGATGGTGAACAGGTTGAACCGATCGCGCGTAAACAGCTGGCGGATCTGGGTATCGATTACTTCGATCTGTACATCGTTCACTTCCCGGTTTCTCTGGATGTTCGTTACCCGCCGTCTTGGCAGAACACCGAAGGTAAAATCAAACTGGGTAAAGCGACCATCCAGGAAACCTGGACCGCGATGGAATCTCTGGTTGAAAAAAAACTGGCGCGTTCTATCGGTGTTTCTAACTTCTCTCCGCAGCTGCTGATGGATCTGCTGCGTTACGCGCGTATCCGTCCGGCGACCCTGCAGATCGAACACCACCCGTACCTGACCCAGAAAACCCTGGTTAACTACGCGCAGGAAGAAGGTATCGCGGTTACCGCGTACTCTTCTTTCGGTCCGCTGTCTTTCATCGAACTGGAAGTTAAAAACGCGCAGAAAACCCCGCGTCTGTTCGATCACGCGGCGATCACCTCTCTGGCGTCTAAATACAACCGTACCCCGGCGCAGGTTCTGCTGCGTTGGGCGACCCAGCGTGGTGTTGCGGTTATCCCGAAATCTAACGATCCGACCCGTCTGGCGCAGAACCTGCAGGTTACCGATTTCGATCTGGAAGCGGATGAAATCGAATCTATCTCTGCGCTGAACCAGAACCTGCGTTTCAACGATCCGCTGAACTACGGTCTGGGTATCACCATCTTCTAA SEQ ID NO: 2: MSSPVVKLSSGYEMPLVGFGLWKVNNDTCADQVYSAIKAGYRLFDGACDYGNEVEVGQGVARAIKEGIVKREDLFLVSKLWNSFHDGEQVEPIARKQLADLGIDYFDLYIVHFPVSLDVRYPPSWQNTEGKIKLGKATIQETWTAMESLVEKKLARS IGVSNFSPQLLMDLLRYARIRPATLQIEHHPYLTQKTLVNYAQEEGIAVTAYSSFGPLSFIELEVKNAQKTPRLFDHAAITSLASKYNRTPAQVLLRWATQRGVAVIPKSNDPTRLAQNLQVTDFDLEADEIESISALNQNLRFNDPLNYGLGITIF The beneficial effects of the present invention include, but are not limited to, the following: This invention uses a semi-rational design method to Penicillium daleae Xylose reductase was modified to obtain a novel xylose reductase mutant with high selectivity for xylose. Using a mixed lignin hydrolysate as a substrate, it catalyzes a reduction reaction to produce xylitol. This invention improves the selectivity of xylose reductase in converting xylose to xylitol by mutating the substrate recognition site. The xylose reductase mutant provided by this invention can be applied to the bioconversion of xylitol using mixed sugar substrates and hemicellulose hydrolysate as raw materials. It eliminates the need for substrate separation and purification. Its excellent xylose selectivity simplifies downstream separation steps in the bioconversion of xylitol, reduces production costs, and improves the purity of the xylitol product. Attached Figure Description

[0041] Figure 1 This demonstrates the reaction mechanism by which xylose reductase catalyzes the formation of xylitol from xylose. Detailed Implementation

[0042] Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific gene cloning operations, please refer to "Molecular Cloning: A Laboratory Manual" edited by J. Sambrook et al.

[0043] Reagents used in upstream genetic engineering operations: The restriction endonucleases, Primer STAR DNA polymerase, DNA ligase, and recombinase used in the embodiments of this invention were all purchased from TaKaRa; the genome extraction kit, plasmid extraction kit, and DNA recovery and purification kit were purchased from Axygen. E. coliBL21 (DE3) and plasmids were purchased from Novagen; DNA markers, low molecular weight standard proteins, and agarose gel electrophoresis reagents were purchased from Beijing TransGen Biotech Co., Ltd.; primer synthesis and gene sequencing were performed by Hangzhou Qingke Zixi Biotechnology Co., Ltd. Please refer to the product instructions for the usage of the above reagents.

[0044] Example 1: Construction of wild-type enzyme engineered bacteria The National Coalition Building Institute (NCBI) database was searched for keywords such as D-xylosereductase, and the amino acid sequence encoding xylose reductase (Sequence ID: XP_056764444.1) was selected. Based on the codon preference of *E. coli*, the amino acid sequence was converted into a nucleotide sequence (i.e., SEQ ID NO:1), and the gene was synthesized and integrated into the multiple cloning site of the expression vector pET-28a. No I and Not Between I and II. Finally, the constructed plasmid was introduced into E. coli BL21(DE3) to construct an engineered strain of wild-type xylose reductase.

[0045] Example 2: Construction of mutant enzyme I. Activation of engineered bacteria and plasmid extraction All engineered bacteria (obtained in Example 1) were activated and cultured using LB medium with the following formulation: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl, dissolved in deionized water and brought to a final volume. The culture was then sterilized at 121°C for 20 min and set aside for use. The solid culture medium was LB medium with 2% agar added.

[0046] The preserved engineered bacterial glycerol tubes were inoculated into test tubes containing 10 mL of LB medium and cultured at 37°C and 220 rpm for 12 h. After obtaining the cultured bacterial cells, plasmids were extracted according to the instructions of the Axygen plasmid extraction kit. The obtained plasmids can be used directly for point mutagenesis or stored long-term at -20°C.

[0047] II. Site-directed gene mutation Gene mutations were obtained using whole-plasmid PCR. Single-site site-directed mutagenesis primers were designed using the online tool PrimerX (www.bioinformatics.org / primerx / cgi-bin / protein_3.cgi), and usage instructions can be found on the website.

[0048] PCR amplification system: DNA polymerase 25 μL 1 μL of upstream primer 1 μL of downstream primer 1 μL of plasmid template ddH2O22 μL PCR amplification conditions: 1) Pre-denaturation: 98℃ for 5 min; 2) Denaturation: 98℃ for 30 s; Annealing: 60℃ for 30 s; Extension: 72℃ for 90 s; 30 cycles in total; 3) Post-extension: 72℃ for 10 min; 4) Store at 4℃.

[0049] After PCR amplification, the amplification products were detected by 1% agarose gel electrophoresis. The results showed that the amplification products were single bands, each approximately 6000 bp in size. The amplification products were then purified and recovered using a DNA purification kit; the specific steps were described in the kit's instructions.

[0050] III. Construction of mutant engineered bacteria Use the purified gene fragment Dpn The wild-type pET28a plasmid template was removed by digestion with an I restriction enzyme (Takara, Code No. 1235A). Then, the digested linear plasmid was recombined with the amplification product using a recombinase to obtain a circular plasmid. The recombinant product (i.e., the circular plasmid) was transformed into... E. coli BL21(DE3) competent cells were plated, and single colonies were picked and cultured in 96-well plates containing LB medium. Enzyme activity and selectivity were measured for unknown mutation sites. Positive mutants were cultured in test tubes containing 10 mL of LB medium to further verify whether selectivity was improved. Mutants with improved selectivity were sequenced for verification. After confirmation, sterile glycerol was added to a final concentration of 25%, and the cells were numbered and stored at -80℃ for later use.

[0051] IV. Construction of multi-point mutant engineered bacteria The plasmids of the best positive mutants from each round were extracted as templates. Based on the Iterative Saturation Mutation (ISM) strategy, saturation mutant libraries of new key amino acid sites were constructed, plated, and cultured according to the construction protocol of the mutant engineered bacteria in Example 2. Similarly, the screened transformants were re-screened and verified by enzyme activity and selectivity assays, and then sequenced for verification. For mutants that passed verification, sterile glycerol at a final concentration of 25% was added, numbered, and stored at -80℃ for later use.

[0052] Example 3: Cultivation of bacterial cells and preparation of crude enzyme solution I. Culture of mutant bacteria LB liquid culture medium composition: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in deionized water and brought to a final volume, sterilized at 121℃ for 20 min, ready for use.

[0053] Single colonies from transformation plates containing the xylose reductase mutant were inoculated into 0.3 mL of LB liquid medium in 96-well plates containing 50 μg / mL kanamycin and cultured at 37°C with shaking for 12 h. Then, 2% of the culture was transferred to 0.5 mL of fresh LB liquid 96-well plates containing 50 μg / mL kanamycin and cultured at 37°C with shaking until OD reached [value missing]. 600 When the concentration reaches approximately 0.6, add IPTG to a final concentration of 0.5 mM, and induce culture at 18°C ​​for 16 h. After culture, centrifuge the culture medium at 4000 rpm for 10 min, discard the supernatant, collect the bacterial cells, and store in an ultra-low temperature freezer at -80°C.

[0054] II. Preparation of crude enzyme solution After the culture was completed, the bacterial cells were collected and washed twice with 100 mM pH 7.5 phosphate buffer. Then, the bacterial cells were resuspended in 100 μL pH 7.5 phosphate buffer and repeatedly frozen and thawed at -80℃ 2-3 times. 100 μL of lysozyme solution with a final concentration of 1.5 g / L was added to resuspend the cells, and the mixture was shaken at 37℃ for 3 h to obtain the crude xylose reductase enzyme solution.

[0055] Example 4: Determination of Xylose Reductase Activity and Selectivity Determination of mutant enzyme activity and selectivity: Xylose reductase activity was determined spectrophotometrically at a detection wavelength of 340 nm, based on the oxidation level of NADPH at 30°C. Xylose reductase activity was measured separately for the substrates xylose and arabinose using a standard reaction system of 200 μL (pH 7.5), containing 200 μM NADPH, 50 mM substrate (xylose or arabinose), 100 mM phosphate buffer, and an appropriate amount of enzyme solution (the volume of crude enzyme solution was adjusted according to enzyme activity, generally 20–80 μL). The enzyme activities of xylose reductase catalyzing xylose and arabinose were calculated using the following formula, and mutant selectivity was obtained.

[0056]

[0057] k1: Slope of the NADPH standard curve k2: Slope of the curve showing the change in absorbance of the reaction sample over time (per minute) V: Total volume of the reaction system v: Volume of added enzyme solution X: Crude enzyme solution dilution factor Enzyme activity is defined as: 1 unit (U) is the amount of enzyme required to consume 1 mmol of NADPH in 1 min under reaction conditions at 30℃. Unit: U / L.

[0058] Method for obtaining the slope (k1) of the NADPH standard curve: Prepare NADPH standard solutions with different concentration gradients, and detect the absorbance at 340 nm using a spectrophotometer. Plot the NADPH standard curve with concentration as the x-axis and absorbance as the y-axis, and obtain the slope through linear fitting.

[0059] Method for obtaining the slope (k2) of the absorbance curve of the reaction sample changing with time (per minute): After the reaction system is prepared, it is placed in a spectrophotometer for detection. Each detection is 1 minute apart, and the corresponding absorbance data is recorded. The reaction progress curve is plotted with reaction time as the x-axis and absorbance value as the y-axis, and the slope is obtained by linear fitting.

[0060]

[0061] Table 1 Results of single-point mutations in the amino acid pocket of xylose reductase substrates

[0062] As shown in Table 1, by saturating the amino acids near the substrate pocket of xylose reductase, several potential amino acid mutation sites were found to improve the selectivity of xylose reductase for xylose substrates. Among them, the five single-point mutations F19G, C48V, K79A, A208L, and R300C had the greatest impact on the selectivity of xylose reductase.

[0063] Table 2 Catalytic activity and selectivity of xylose reductase and its mutants

[0064] As shown in Table 2, after combinatorial active site saturation mutation... A novel semi-rational design method using the sitesaturation test (CAST) and iterative saturation mutagenesis (ISM) screened 14 mutant strains as shown in Table 2. This method showed significantly improved selectivity compared to the wild type (SEQ ID NO: 2), but due to the existence of trade... The off phenomenon also resulted in a certain degree of decrease in xylanase activity in some mutants. Even so, most mutants still exhibited advantages in xylanase activity and selectivity compared to the wild type.

[0065] Example 5: Whole-cell catalytic mixed substrate According to a particularly preferred embodiment of the present invention, a method for the whole-cell catalytic production of xylitol is provided. Recombinant bacterial strain whole cells are used as the biocatalyst. The reaction system consists of 10 g / L glucose, 20 g / L lignin hydrolysate, and 5 g / L bacterial cells. The pH of the reaction system is controlled at 7.5, and the reaction is carried out at 220 rpm at 30°C to achieve intracellular catalytic reaction for the preparation of xylitol. The substrate is xylose with an initial concentration of 500 mM, and 200 mM glucose is added simultaneously. During the reaction, the xylose content is maintained between 1% and 1.5%, and ammonia is added as needed to maintain pH stability. The reaction solution is treated in a water bath at 80°C for 20 minutes to terminate the reaction. Samples can then be prepared and analyzed by HPLC.

[0066] Liquid chromatography (LC) detection conditions: Xylose, arabinose, xylitol, and arabinitol were quantitatively detected using Shimadzu LC-20AD. The detector was a Shimadzu RID-20A differential refractive index detector; the column was a Rezex ROA organic acid H+ (8%) column (Phenomenex, USA); the mobile phase was 5 mmol sulfuric acid; the flow rate was set to 0.4 mL / min; and the column temperature was set to 60℃.

[0067]

[0068] Table 3. Selectivity and conversion rate of xylose reductase and its mutants

[0069] The results show that a mutant enzyme with significantly improved selectivity for xylose was obtained through targeted molecular modification of XR21 xylose reductase. This mutant can efficiently catalyze the reduction reaction to xylitol using mixed lignin hydrolysate as a substrate. Compared with the wild-type enzyme (SEQ ID NO: 2), it significantly reduced the catalytic activity for the epimeric arabinose. This characteristic eliminates the need for pretreatment and purification of the mixed sugar substrate during xylitol production, effectively simplifying the downstream separation process, reducing production costs, and significantly improving the purity of the xylitol product. This invention provides a new, efficient, economical, and environmentally friendly biocatalytic production route for xylitol suitable for industrial applications, with broad application prospects.

Claims

1. A xylose reductase mutant, characterized in that, The mutant has five or fewer substitutions relative to SEQ ID NO: 2 and has less than 100% sequence identity with SEQ ID NO: 2, wherein the substitutions are selected from: F19A, F19L, F19I, F19M, F19G, F19S, F19T, F19Y, F19H, F19E, C48A, C48V, C48S, K79A, K79M, K79N, K79R, A208L, A208W, A208G, A208Y, A208D, R300M, R300T, R300C, R300Q, and R300E, or combinations thereof.

2. The xylose reductase mutant according to claim 1, characterized in that, The substituted material is selected from: F19G, C48V, K79A, A208L and R300C, or a combination thereof.

3. A xylose reductase mutant, characterized in that, The mutant was obtained by performing any of the following mutations on SEQ ID NO: 2: F19G; C48V; K79A; A208L; R300C; F19G+K79A; C48V+A208L; A208L+R300C; F19G+C48V+A208L; F19G+K79A+R300C; C48V+K79A+R300C; F19G+C48V+K79A+A208L; C48V+K79A+A208L+R300C; F19G+C48V+K79A+A208L+R300C; F19A; F19L; F19I; F19M; F19S; F19T; F19Y; F19H; F19E; C48A; C48S; K79M; K79N; K79R; A208W; A208G; A208Y; A208D; R300M; R300T; R300Q or R300E.

4. A polynucleotide, characterized in that, Encode the xylose reductase mutant according to any one of claims 1-3.

5. A recombinant vector, characterized in that, It contains the polynucleotide as described in claim 4.

6. A host cell, characterized in that, It contains the polynucleotide according to claim 4 or the recombinant vector according to claim 5.

7. The host cell according to claim 6, characterized in that, The host cell is a fungal cell, a bacterial cell, or a plant cell. Preferably, the host cell is a bacterial cell, and more preferably, the bacterial cell is an Escherichia coli cell.

8. The use of the xylose reductase mutant according to any one of claims 1-3, the polynucleotide according to claim 4, the recombinant vector according to claim 5, or the host cell according to claim 6 or 7 in the production of xylitol.

9. A method for producing a xylose reductase mutant according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Culture the host cells according to claim 6 or 7 under suitable conditions for expressing the xylose reductase mutant; and (2) The xylose reductase mutant was recovered.

10. A method for preparing xylitol, characterized in that, The method comprises: mixing xylose and NADPH with a xylose mutant according to any one of claims 1-3 or a host cell according to claim 6 or 7 under suitable conditions to generate xylose.

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